Endocrine cells in the pancreas synthesize and secrete hormones required for energy homeostasis and nutrient metabolism. In particular, insulin-producing beta cells are required to control systemic blood glucose levels, and their loss or dysfunction leads to diabetes. The endocrine cells that make up pancreatic islets form during development from the differentiation of endocrine precursors, or pro-endocrine cells. Although all five endocrine lineages derive from a common pool of endocrine precursors, how these pro-endocrine cells become committed to a particular endocrine cell subtype is poorly understood. A more detailed molecular characterization of the precise stages of differentiation from an endocrine precursor to a differentiated endocrine cell is needed. During pancreatic organogenesis, a subset of cells in the ductal epithelium transiently expresses Neurogenin3 (Ngn3), a pro-endocrine transcription factor that is required for endocrine lineage specification. Lineage tracing has demonstrated that all five endocrine lineages are derived from these Ngn3-expressing precursors, and that Ngn3-expressing cells do not give rise to other pancreatic lineages besides endocrine. It is not yet determined if each Ngn3(+) cell is pre-committed to a specific endocrine lineage or if cell fate decisions are made at a later stage, after Ngn3 expression. Using single-cell RNA sequencing (RNA-seq) of murine pancreata, our laboratory has identified a novel cell population that expresses endocrine lineage genes and is defined by differential expression of the transcription factor Fev. This FevHI population is also characterized by lack of expression of Ngn3(+) or of the pancreatic hormones detected in differentiated endocrine cells. Lineage tracing of Ngn3-expressing cells in vivo has revealed that Fev-expressing cells are descendants of Ngn3(+) pro-endocrine cells. Thus, this novel putative endocrine precursor population appears to represent an intermediate stage following transient Ngn3 expression and before hormone acquisition. The experiments outlined in this proposal begin with a focus on characterizing the spatial and temporal appearance of this novel FevHI population in human fetal and adult pancreata. In addition, genetic lineage tracing studies will be undertaken to define the downstream lineage of this putative FevHI intermediate precursor. Lastly, as Fev whole body knockout mice display defects in glucose homeostasis, the role of FEV itself in endocrine cell specification and functional maturation of human beta cells will be investigated using a human embryonic stem cell-based platform and genomic engineering strategies. The overarching goal of these studies is both to provide new insights into the basic biology underlying endocrine cell fate allocation, as well as to harness this knowledge for generating functional human embryonic stem cell-derived beta cells and regenerating endogenous beta cells for the treatment of diabetes.